Fibroblast growth factor antagonists

ABSTRACT

Antagonists to basic fibroblast growth factor, a 146 amino acid residue polypeptide, are produced. These antagonists are generally between 4 and 45 residues in length and are characterized by their ability to interact with the FGF receptor and/or inhibit and therefore modulate endothelial and other cell growth. One group of these antagonists includes the four residue sequence which forms basic FGF(36-39), namely Pro-Asp-Gly-Arg. Another of these antagonists includes the sequence of bovine basic FGF(106-115), namely Tyr-Arg-Ser-Arg-Lys-Tyr-Ser-Ser-Trp-Tyr. These peptides are also antagonistic to acidic FGF and other members of the family of FGF peptides. They are effective to combat FGF-promoted mitosis in melanomas and the like.

This invention was made with Government support under Grant Nos.HD-09690 and AM-18881, awarded by the National Institutes of Health. TheGovernment has certain rights in this invention.

This application is a continuation-in-part of earlier application Ser.No. 854,843, filed Apr. 22, 1986, now abandoned.

The present invention is directed to fibroblast growth factor (FGF) andmore particularly to FGF antagonists produced by synthetic methods,which can be used to reduce the effects of mammalian FGF in certaininstances.

BACKGROUND OF THE INVENTION

Both the brain and the pituitary gland have been known to containmitogenic factors for cultured cells; however, until 1974, it wasunclear what their relationship was with classical pituitary hormones,such as TSH, LH, FSH, GH and ACTH. In 1974, the purification of a bovinegrowth factor called basic fibroblast growth factor (bFGF) was reportedwhich was shown to from pituitary hormones, Gospodarowicz, D. Nature.249, 123-127 (1974) This growth factor is now known to have a MW of16,415, is basic (a pI of 9.6), and is a potent mitogen for eitherdistinct normal diploid fibroblasts or established cell lines.Purification of another growth factor, acidic brain fibroblast growthfactor (aFGF) is described in U.S. Pat. No. 4,444,760 (Apr. 24, 1984).Complete characterization of bovine aFGF was reported by Esch et al.,Biochemical and Biophysical Research Communications, 133, 554-562(1985).

Later studies confirmed that, in addition to fibroblasts, FGF is alsomitogenic for a wide variety of normal diploid mesoderm-derived andneural crest-derived cells including granulosa cells, adrenal corticalcells, chondrocytes, myoblasts, corneal and vascular endothelial cellsfrom either bovine or human origin, vascular smooth muscle cells, thelens epithelial cells. FGF has also been shown to substitute forepithelial cells. FGF has also been shown to substitute forplatelet-derived growth factor in its ability to support theproliferation of fibroblasts exposed to plasma-supplemented medium.Consistent with its ability to stimulate the proliferation of bovine andhuman vascular endothelial cells, FGF has a similar activity in vivoupon capillary endothelial cells; therefore, FGF is considered anangiogenic factor.

SUMMARY OF THE INVENTION

The present invention provides FGF antagonists which may be produced bysynthetic methods and which substantially counteract the biologicaleffect of mammalian FGF in certain instances.

The present invention provides antagonists to basic and acidicfibroblast growth factor which may be synthesized using recombinant DNAtechniques or other suitable techniques, such as classical or solidphase synthesis. Basic FGF is a 146 amino acid residue polypeptidehaving the sequence set forth hereinafter. It appears most likely that,in the native bovine FGF molecule, none of the cysteine residues aredisulfide-bonded to each other, but that there may be bonding of one ormore of the cysteine residues to free cysteine molecules. In any case,the present invention provides biologically active peptides thatsuppress the biological activity of FGF. They can be synthesized by arecombinant DNA technique or by standard chain elongation proceduresinvolving stepwise addition of amino acid residues, such as solid-phasesynthesis upon a solid resin support.

Pharmaceutical compositions in accordance with invention include FGFantagonists or nontoxic salts thereof dispersed in a pharmaceuticallyacceptable liquid or solid carrier. Such pharmaceutical compositions canbe used in clinical medicine, both human and veterinary, and in acute orchronic administration for diagnostic or therapeutic purposes. They areuseful both in vivo and in vitro in modulating the growth of endothelialand other related cell types.

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS

The invention provides antagonists to mammalian FGF, particularly tobovine basic FGF, but also to acidic FGF, which can be readilysynthesized. The nomenclature used to define the peptides is thatspecified by Schroder & Lubke, "The Peptides", Academic Press (1965),wherein in accordance with conventional representation the residuehaving the free alpha-amino group at the N-terminus appears to left andthe residue having the alpha-carboxyl group at the C-terminus to theright. Where the amino acid residue has isomeric forms, it is the L-formof the amino acid that is represented. Bovine basic FGF has been foundto be a peptide having the following sequence: ##STR1## The C-terminusof the native molecule is free acid.

The present invention provides two families of FGF antagonists which areeach based upon a central fragment from the native hormone bFGF. Thecore of the first is those residues appearing at positions 36-39, andthe core of the second appears to be those residues appearing atpositions 106-115. In other words, relatively short peptides containingthe four residues of the first family, as well as the tetrapeptideitself, show some suppression of endothelial cell growth, when growingunder nonstimulated conditions (serum alone) and also when serum issupplemented by the addition of FGF to in vitro cell cultures. The FGFantagonism of the first family is found to be very substantiallyincreased by the inclusion of N-terminal and/or C-terminal extensions tothe tetrapeptide. These extensions may comprise the residue sequencesnormally found at these locations in the native hormone, e.g.,bFGF(30-50) and are preferably, but not necessarily, amidated at theC-terminus. Preferably, the extended fragment includes bFGF(24-68) whichexhibits good FGF antagonism. Some substitutions may be made in thesequence at selected locations, as discussed hereinafter.

The basis for the antagonistic action exhibited by these peptides is aninteraction with the FGF receptor. Peptides that show antagonism tomitogenesis in vitro (including all FGF target cell types) also preventFGF from binding to its receptor, and it appears the minimum lengthpeptide should contain either the core sequence of bFGF (36-39) or bFGF(106-115).

In the peptidic fragments of the second family, which are generallywithin the sequence of bFGF (93-120) and which are also antagonistic,there is also a distinct heparin-binding site, i.e., a sequencecontained within the peptide fragment binds radioactive heparin as wellas the receptor. Because heparin is an important element in certain FGFaction, peptides that inhibit binding between FGF and heparin may wellalso exhibit the important capacity to inhibit the biological action ofFGF, which may be an inhibition of the binding of FGF to its receptorresulting from this interaction between FGF and heparin. However, itappears that it may be possible to design FGF antagonists that will bindstrongly to the receptor and not bind strongly to heparin; for example,by replacing certain of the residues that account for binding toheparin, e.g., those in positions 107-110, analogs should result whichwill not bind heparin without substantially detracting from binding tothe FGF receptor. The specificity of fragments related to bFGF(24-68)and bFGF(93-120) is best illustrated by a) their effects on all threeparameters of FGF action (i.e., mitogenesis, heparin-binding andreceptor interaction) and b) the observation that other FGF peptidefragments which do not contain either of the afore-mentioned coresequences fail to exhibit similar activity.

The first family of FGF antagonist peptides provided by the inventionmay be expressed by the following formula (which is based upon thenaturally occurring sequence of bovine bFGF):Tyr-Cys-Lys-Asn-Gly-Gly-Phe-Phe-Leu-Arg-Ile-His-Pro-Asp-Gly-Arg-Val-Asp-R.sub.42-Val-Arg-Glu-Lys-R₄₇-Asp-Pro-His-Ile-Lys-Leu-Gln-Leu-Gln-Ala-Glu-Glu-Arg-Gly-Val-Val-Ser-Ile-Lys-Gly-Val-Ywherein Y is either OH or NH₂. R₄₂ may be Gly or Ala or Sar, and R₄₇ maybe Ser or Ala or Thr. Sar is the abbreviation for sarcosine. Peptideshaving this entire length, i.e., 45 residues, function as FGFantagonists and not as partial agonists. As such, they suppressendothelial cell growth both in the presence of basal FGF as well as inthe presence of added FGF. 45 residues is not considered to be a maximumlimit for a peptide that will function as an FGF antagonist, a mainfunction of such an antagonist being simply to block the receptor on theendothelial cells without causing activation. As a result, additionalresidues may be added to either or both termini so long as the presenceof these additional residues does not either (a) turn the peptide into apartial FGF agonist or (b) detract from the binding of the peptide tothe receptor so as to lessen its biological activity as an FGFantagonist.

The second family of FGF antagonist peptides provided by the inventionmay be expressed by the following formula (which is based on thenaturally occurring sequence of bovine bFGF):Phe-Phe-Phe-Glu-Arg-Leu-Glu-Ser-Asn-Asn-Tyr-Asn-Thr-Tyr-Arg-Ser-Arg-Lys-Tyr-R₁₁₂-R₁₁₃ -R₁₁₄ -R₁₁₅ -Val-Ala-Leu-Lys-Arg-Y, wherein one or more of theresidues in the sequence: Tyr-Arg-Ser-Arg-Lys-Tyr can be substituted byits D-isomer, R₁₁₂ is Ser, Thr or D-Ser, R₁₁₃ is Ser, Ala or D-Ser, R₁₁₄is Trp or Met, R₁₁₅ is Tyr or Phe, and Y is either OH or NH₂. Thesepeptides function as FGF antagonists, suppressing endothelial cellgrowth (and growth in other FGF target cells) in the presence or absenceof FGF, and peptides shorter in length which include the core sequencebFGF(106-115) are also effective to act as antagonists to FGF. Althoughvarious of these peptides may also exhibit some agonist activity, ifresidues are be added to either or both termini of this 28-residuesequence, such changes may result in some receptor activation, creatingcompetitive antagonists having still greater agonist activity.

It may be preferable to synthesize peptides which are about 45 aminoacids or greater in length by using recombinant DNA methods. On theother hand, it may be preferable to synthesize peptides of about 30residues or less in length using the well-known chain elongationtechniques, such as solid-phase synthesis, as on a Merrifield resin orthe like.

To synthesize a bFGF peptide containing only naturally occurring aminoacid residues by recombinant DNA, a double-stranded DNA chain whichencodes the desired amino acid sequence is synthetically constructed.The degeneracy of the genetic code permits a wide variety of codoncombinations to be used to form the DNA chain that encodes the productpolypeptide. Certain particular codons are more efficient forpolypeptide expression in certain types of organisms, and the selectionof codons preferably is made according to those codons which are mostefficient for expression in the type of organism which is to serve asthe host for the recombinant vector. However, any correct set of codonsshould encode the desired product, even if slightly less efficiently.Codon selection may also depend upon vector construction considerations;for example, it may be necessary to avoid creating a particularrestriction site in the DNA chain if, subsequent to insertion of thesynthetic DNA chain, the vector is to be manipulated using a restrictionenzyme that cleaves at such a site. Also, it is necessary to avoidplacing restriction sites in the DNA chain if the host organism which isto be transformed with the recombinant vector containing the DNA chainis known to produce a restriction enzyme that would cleave at such asite within the DNA chain.

In addition to the bFGF antagonist-encoding sequences, the DNA chainthat is synthesized may contain additional sequences, depending uponvector construction considerations. Typically, a DNA chain issynthesized with linkers at its ends to facilitate insertion intorestriction sites within a cloning vector. The DNA chain may beconstructed so as to encode the desired sequence as a portion of afusion polypeptide; and if so, it will generally contain terminalsequences that encode amino acid residue sequences that serve asproteolytic processing sites, whereby the desired polypeptide may beproteolytically cleaved from the remainder of the fusion polypeptide.The terminal portions of the synthetic DNA chain may also containappropriate start and stop signals.

To assemble the desired DNA chain, oligonucleotides are constructed byconventional methods, such as procedures described in T. Maniatis etal., Cold Spring Harbor Laboratory Manual. Cold Spring Harbor, N.Y.(1982)(hereinafter, CSH). Sense and antisense oligonucleotide chains, upto about 70 nucleotide residues long, are synthesized, preferably onautomated synthesizers, such as the Applied Biosystem Inc. model 380ADNA synthesizer. The oligonucleotide chains are constructed so thatportions of the sense and antisense oligonucleotides overlap,associating with each other through hydrogen bonding betweencomplementary base pairs and thereby forming double stranded chains, inmost cases with gaps in the strands. Subsequently, the gaps in thestrands are filled in and oligonucleotides of each strand are joined endto end with nucleotide triphosphates in the presence of appropriate DNApolymerases and/or with ligases.

As an alternative to construction of a synthetic DNA chain througholigonucleotide synthesis, when a peptide is desired that is a segmentof the naturally occurring molecule, cDNA corresponding to the desiredbFGF fragment may be prepared. A cDNA library or an expression libraryis produced in a conventional manner by reverse transcription frommessenger RNA (mRNA) from a bFGF-producing cell line. To select clonescontaining bFGF sequences, hybridization probes (preferably mixed probesto accommodate the degeneracy of the genetic code) corresponding toportions of the bFGF protein are produced and used to identify clonescontaining such sequences. Screening of the expression library with bFGFantibodies may also be used, alone or in conjunction with hybridizationprobing, to identify or confirm the presence of bFGF-encoding DNAsequences in DNA library clones. Such techniques are taught, for examplein CSH,supra.

The double-stranded bFGF-encoding DNA chain is shortened appropriatelyto the desired length to create the peptide of interest and thenmodified as necessary to permit its insertion into a particularappropriate cloning vector in mind. The cloning vector that is to berecombined to incorporate the DNA chain is selected appropriate to itsviability and expression in a host organism or cell line, and the mannerof insertion of the DNA chain depends upon factors particular to thehost. For example, if the DNA chain is to be inserted into a vector forinsertion into a prokaryotic cell, such as E. Coli, the DNA chain willbe inserted 3' of a promoter sequence, a Shine-Delgarno sequence (orribosome binding site) that is within a 5' non-translated portion and anATG start codon. The ATG start codon is appropriately spaced from theShine-Delgarno sequence, and the encoding sequence is placed in correctreading frame with the ATG start codon. The cloning vector also providesa 3' non-translated region and a translation termination site. Forinsertion into a eukaryotic cell, such as a yeast cell or a cell lineobtained from a higher animal, the FGF fragment-encoding oligonucleotidesequence is appropriately spaced from a capping site and in correctreading frame with an ATG start signal. The cloning vector also providesa 3' non-translated region and a translation termination site.

Prokaryotic transformation vectors, such as pBR322, pMB9, Col E1, pCRI,RP4 and lambda-phage, are available for inserting a DNA chain of thelength necessary to encode the FGF fragments of interest withsubstantial assurance of at least some expression of the encodedpolypeptide. Typically, such vectors are constructed or modified to havea unique restriction site(s) appropriately positioned relative to apromoter, such as the lac promoter. The DNA chain may be inserted withappropriate linkers into such a restriction site, with substantialassurance of production of FGF in a prokaryotic cell line transformedwith the recombinant vector. To assure the proper reading frame, linkersof various lengths may be provided at the ends of the FGFpeptide-encoding sequence. Alternatively, cassettes, which includesequences, such as the 5' region of the lac Z gene (including theoperator, promoter, transcription start site, Shine Delgarno sequenceand translation initiation signal), the regulatory region from thetryptophane gene (trp operator, promoter, ribosome binding site andtranslation initiator), and a fusion gene containing these twopromoters, called the trp-lac or commonly called the Tac promoter, areavailable into which a synthetic DNA chain may be conveniently insertedbefore the cassette is inserted into a cloning vector of choice.

Similarly, eukaryotic transformation vectors, such as the cloned bovinepapilloma virus genome, the cloned genomes of the murine retroviruses,and eukaryotic cassettes, such as the pSV-2 gpt system (described byMulligan and Berg, Nature 277, 108-114, 1979), the Okayama-Berg cloningsystem (Mol. Cell Biol. 2, 161-170, 1982) and the expression cloningvector recently described by Genetics Institute (Science 228, 810-815,1985), are available which provide substantial assurance of at leastsome expression of the FGF peptide in the transformed eukaryotic cellline.

Another way to produce bFGF fragments of desired length is to producethe polypeptide initially as a segment of a gene-encoded fusionpolypeptide. In such case, the DNA chain is constructed so that theexpressed polypeptide has enzymatic processing sites flanking the bFGFfragment sequence. A bFGF-fragment-encoding DNA chain may be inserted,for example, into the beta-galactosidase gene for insertion into E.Coli, in which case, the expressed fusion polypeptide is subsequentlycleaved with appropriate proteolytic enzymes to release the bFGFfragment from beta-galactosidase peptide sequences.

An advantage of inserting the bFGF-fragment-encoding sequence so that itis expressed as a cleavable segment of a fusion polypeptide, e.g., asthe bFGF-fragment sequence fused within the beta-galactosidase peptidesequence, is that the endogenous polypeptide into which the bFGFfragment sequence is inserted is generally rendered non-functional,thereby facilitating selection for vectors encoding the fusion peptide.

The peptides can be synthesized by suitable chain elongation orcoupling-type methods, such as by exclusively solid-phase techniques, bypartial solid-phase techniques, by fragment condensation or by classicalsolution couplings. The techniques of exclusively solid-phase synthesisare set forth in the textbook "Solid-Phase Peptide Synthesis", Stewart &Young, Pierce Chemical Co., Rockford, Ill., 1984, and are exemplified bythe disclosure of U.S. Pat. No. 4,105,603, issued Aug. 8, 1978. Thefragment condensation method of synthesis is exemplified in U.S. Pat.No. 3,972,859 (Aug. 3, 1976). Other available syntheses are exemplifiedby U.S. Pat. No. 3,842,067 (Oct. 15, 1974) and U.S. Pat. No. 3,862,925(Jan. 28, 1975).

Common to coupling-type syntheses is the protection of the labileside-chain groups of the various amino acid moieties with suitableprotecting groups which will prevent a chemical reaction from occurringat that site until the group is ultimately removed. Usually also commonis the protection of an alpha-amino group on an amino acid or a fragmentwhile that entity reacts at the carboxyl group, followed by theselective removal of the alpha-amino protecting group to allowsubsequent reaction to take place at that location. Accordingly, it iscommon that, as a step in the synthesis, an intermediate compound isproduced which includes each of the amino acid residues located in itsdesired sequence in the peptide chain with side-chain protecting groupslinked to the appropriate residues.

Such an intermediate for the first family may have the formula: X¹Tyr(X²)-Cys(X⁴)-Lys(X⁷ -Gly-Gly-Phe-Phe-Leu-Arg(X⁶)-Ile-His(X⁹-Pro-Asp(X³)Gly-Arg(X⁶)-Val-Asp(X³)-R₄₂ -Val-Arg(X⁶)-Glu(X³)-Lys(X⁷)-R₄₇(X⁵)-Asp(X³)-Pro-His(X⁹)-Ile-Lys(X⁷-Leu-Gln(X⁸)-Leu-Gln(X⁸)-Ala-Glu(X³)-Glu(X³)-Arg(X.sup.6)-Gly-Val-Val-Ser(X⁵)-Ile-Lys(X⁷)-Gly-Val-X¹⁰.

Such an intermediate for the second family may have the formula: X¹-Phe-Phe-Phe-Glu(X³)-Arg(X⁶)-Leu-Glu(X³)-Ser(X⁵)-Asn(X⁸)-Asn(X⁸)-Tyr(X²)-Asn-Thr(X⁵)-Tyr(X²)-Arg(X⁶)-Ser(X⁵)-Arg(X⁶)-Lys(X.sup.7)-Tyr(X²)-Ser(X⁵)-Ser(X⁵)-Trp-Tyr(X²)-Val-Ala-Leu-Lys(X⁷)-Arg(X⁶)-X¹⁰.

In these formulae: X¹ is either hydrogen or an alpha-amino protectinggroup. The alpha-amino protecting groups contemplated by X¹ are thoseknown to be useful in the art of step-wise synthesis of polypeptides.Among the classes of alpha-amino protecting groups covered by X¹ are (1)acyl-type protecting groups, such as formyl, trifluoroacetyl, phthalyl,toluenesulfonyl(Tos), benzensulfonyl, nitrophenylsulfenyl,tritylsulfenyl, o-nitrophenoxyacetyl, chloroacetyl, acetyl, andγ-chlorobutyryl; (2) aromatic urethan-type protecting groups, such asbenzyloxycarbonyl(Z) and substituted Z, such asp-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl; (3) aliphaticurethan protecting groups, such as t-butyloxycarbonyl (BOC),diisopropylmethyloxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl,allyloxycarbonyl; (4) cycloalkyl urethan-type protecting groups, such ascyclopentyloxycarbonyl, adamantyloxycarbonyl,and cyclohexyloxycarbonyl;(5) thiourethan-type protecting groups, such as phenylthiocarbonyl; (6)alkyl-type protecting groups, such as triphenylmethyl (trityl),benzyl;(7) trialkylsilane groups, such as trimethylsilane. The preferredalpha-amino protecting group is BOC.

X² is a protecting group for the phenolic hydroxyl group of Tyr selectedfrom the group consisting of tetrahydropyranyl, tert-butyl, trityl, Bzl,CBZ, Br-CBZ and 2,6-dichlorobenzyl. The preferred protecting group is2,6-dichlorobenzyl. X² can be hydrogen which means that there is noprotecting group on the hydroxyl group.

X³ is hydrogen or an ester-forming protecting group for the carboxylgroup of Asp or Glu and is selected from the group consisting of Bzl,cyclohexyl, cycloheptal, 2,6-dichlorobenzyl, methyl and ethyl.

X⁴ is a protecting group for Cys selected from the group consisting ofp-methoxy-benzyl(MeOBzl), p-methylbenzyl, acetamidomethyl, trityl andBzl. The most preferred protecting group is p-methoxybenzyl. X⁴ can alsobe hydrogen, meaning that there is no protecting group on thesulfhydryl.

X⁵ is a protecting group for the hydroxyl group of Thr and Ser and isselected from the group consisting of acetyl, benzoyl, tert-butyl,trityl, tetrahydropyranyl, Bzl, 2,6-dichlorobenzyl and CBZ. Thepreferred protecting group is Bzl. X⁵ can be hydrogen, which means thereis no protecting group on the hydroxyl group.

X⁶ is a protecting group for the guanido group of Arg selected from thegroup consisting of nitro, Tos, CBZ, adamantyloxycarbonyl, and BOC, oris hydrogen.

X⁷ is hydrogen or a protecting group for the side-chain aminosubstituent of Lys. Illustrative of suitable side-chain amino protectinggroups are 2-chlorobenzyloxycarbonyl(2-Cl-Z), Tos, CBZ,t-amyloxycarbonyl and BOC.

The selection of a side-chain amino protecting group is not criticalexcept that it must be one which is not removed during deprotection ofthe alpha-amino groups during the synthesis. Hence, the alpha-aminoprotecting group and the side-chain amino protecting group cannot be thesame.

X⁸ is a protecting group for the side-chain amido group of Gln and/orAsn and is preferably xanthyl (Xan). Optionally X⁸ can be hydrogen.

X⁹ is a protecting group for the imidazole nitrogen of His, such as Tosor dinitrophenyl, or may be hydrogen.

X¹⁰ is selected from the class consisting of

OH, OCH₃, esters, amides, hydrazides, --O--CH₂ -resin support and--NH-resin support, with the groups other than OH and amides beingbroadly considered as protecting groups.

In the formula for the intermediate, at least one of X¹, X², X³, X⁴, X⁵,X⁶, X⁷, X⁸, X⁹ and X¹⁰ is a protecting group.

In selecting a particular side-chain protecting group to be used in thesynthesis of the peptides, the following rules are followed: (a) theprotecting group should be stable to the reagent and under the reactionconditions selected for removing the alpha-amino protecting group ateach step of the synthesis, (b) the protecting group should retain itsprotecting properties and not be split off under coupling conditions,and (c) the side-chain protecting group should be removable, upon thecompletion of the synthesis containing the desired amino acid sequence,under reaction conditions that will not alter the peptide chain.

The peptides are preferably prepared using solid phase synthesis, suchas that described by Merrifield, J. Am. Chem. Soc., 85, p 2149 (1963),although other equivalent chemical syntheses known in the art can alsobe used as previously mentioned. Solid-phase synthesis is commenced fromthe C-terminal end of the peptide by coupling a protected alpha-aminoacid to a suitable resin. Such a starting material can be prepared byattaching alpha-amino-protected Val by an ester linkage to achloromethylated resin or a hydroxymethyl resin, or by an amide bond toa BHA resin or MBHA resin. The preparation of the hydroxymethyl resin isdescribed by Bodansky et al., Chem. Ind. (London) 38, 1597-98 (1966).Chloromethylated resins are commercially available from Bio RadLaboratories, Richmond, California and from Lab. Systems, Inc. Thepreparation of such a resin is described by Stewart et al., "Solid PhasePeptide Synthesis"(Freeman & Co., San Francisco 1969), Chapter 1, pp1-6. BHA and MBHA resin supports are commercially available and aregenerally used only when the desired polypeptide being synthesized hasan alpha-carboxamide at the C-terminal.

For example, a peptide of the first family can be prepared by couplingVal, protected by BOC, to a chloromethylated resin according to theprocedure of Monahan and Gilon, Biopolymer 12, pp 2513-19, 1973 when,for example, it is desired to synthesize such a peptide with freecarboxy terminus. Following the coupling of BOC-Val, the alpha-aminoprotecting group is removed, as by using trifluoroacetic acid(TFA) inmethylene chloride, TFA alone or HCl in dioxane. The deprotection iscarried out at a temperature between about 0° C. and room temperature.Other standard cleaving reagents and conditions for removal of specificalpha-amino protecting groups may be used as described in Schroder &Lubke, "The Peptides", 1 pp 72-75 (Academic Press 1965).

After removal of the alpha-amino protecting group of Val, the remainingalpha-amino- and side-chain-protected amino acids are coupled stepwisein the desired order to obtain an intermediate compound as definedhereinbefore. As an alternative to adding each amino acid separately inthe synthesis, some of them may be coupled to one another prior to theiraddition to the solid phase reactor. The selection of an appropriatecoupling reagent is within the skill of the art; particularly suitableas a coupling reagent is N,N'-dicyclohexyl carbodiimide (DCCI).

Activating reagents used in solid phase synthesis of the peptides arewell known in the peptide synthesis art. Examples of suitable activatingreagents are: (1) carbodiimides, such as N,N'-diisopropyl carbodiimide,N-N'-dicyclohexylcarbodiimide(DCCI); (2) cyanamides such asN,N'-dibenzylcyanamide; (3) keteimines; (4) isoxazolium salts, such asN-ethyl-5-phenyl isoxazolium-3,-sulfonate; (5) monocyclicnitrogen-containing heterocyclic amides of aromatic character containingone through four nitrogens in the ring, such as imidazolides,pyrazolides, and 1,2,4-triazolides. Specific heterocyclic amides thatare useful include N,N'-carbonyl diimidazole,N,N'-carbonyl-di-1,2,4-triazole; (6) alkoxylated acetylene, such asethoxyacetylene; (7) reagents which form a mixed anhydride with thecarboxyl moiety of the amino acid, such as ethylchloroformate andisobutylchloroformate and (8) reagents which form an active ester withthe carboxyl moiety of the amino acid, such as nitrogen-containingheterocyclic compounds having a hydroxy group on one ring nitrogen, e.g.N-hydroxyphthalimide, N-hydroxysuccinimide and1-hydroxybenzotriazole(HOBT). Other activating reagents and their use inpeptide coupling are described by Schroder & Lubke supra, in Chapter IIIand by Kapoor, J. Phar. Sci., 59, pp 1-27 (1970).

Each protected amino acid or amino acid sequence is introduced into thesolid phase reactor in about a twofold or more excess, and the couplingmay be carried out in a medium of dimethylforaamide(DMF):CH₂ Cl₂ (1:1)or in DMF or CH₂ Cl₂ alone. In cases where incomplete coupling occurs,the coupling procedure is repeated before removal of the alpha-aminoprotecting group prior to the coupling of the next amino acid. Ifperformed manually, the success of the coupling reaction at each stageof the synthesis is monitored by the ninhydrin reaction, as described byE. Kaiser et al., Anal. Biochem. 34, 595 (1970).

After the desired amino acid sequence has been completed, theintermediate peptide is removed from the resin support by treatment witha reagent, such as liquid hydrogen fluoride, which not only cleaves thepeptide from the resin but also cleaves all remaining side-chainprotecting groups X², X³, X⁴, X⁵, X⁶, X⁷, X⁸ and X⁹ and the alpha-aminoprotecting group X¹ to obtain the peptide.

As an alternative route, the intermediate peptide may be separated fromthe resin support by alcoholysis after which the recovered C-terminalalkyl ester is converted to the acid by hydrolysis. Any side-chainprotecting groups may then be cleaved as previously described or byother known procedures, such as catalytic reduction (e.g. Pd on BaSO₄)When using hydrogen fluoride for cleaving, anisole and methylethylsulfide are included in the reaction vessel for scavenging.

The following examples set forth preferred methods for synthesizing FGFantagonists by the solid-phase technique. It will of course beappreciated that the synthesis of a correspondingly shorter peptidefragment is effected in the same manner by merely eliminating therequisite number of amino acids at either end of the chain.

EXAMPLE I

The synthesis of bFGF(24-68)-amide having the formula:H-Tyr-Cys-Lys-Asn-Gly-Gly-Phe-Phe-Leu-Arg-Ile-His-Pro-Asp-Gly-Arg-Val-Asp-Gly-Val-Arg-Glu-Lys-Ser-Asp-Pro-His-Ile-Lys-Leu-Gln-Leu-Gln-Ala-Glu-Glu-Arg-Gly-Val-Val-Ser-Ile-Lys-Gly-Val-NH₂is conducted in a stepwise manner using a Beckman 990 PeptideSynthesizer and an MBHA resin. Coupling of BOC-Val to the resin isperformed by the general procedure set forth in U.S. Pat. No. 4,292,313,and it results in the substitution of about 0.2-0.6 mmol Val per gram ofresin depending on the substitution of the MHBA resin used.

After deprotection and neutralization, the peptide chain is builtstep-by-step on the resin. Deprotection, neutralization and addition ofeach amino acid is performed in general accordance with the procedureset forth in detail in Guillemin et al. U.S. Pat. No. 3,904,594. Thecouplings are specifically carried out as set out in the followingschedule.

    ______________________________________                                        SCHEDULE                                                                                                    MIX TIMES                                       STEP  REAGENTS AND OPERATIONS MIN.                                            ______________________________________                                         1    CH.sub.2 Cl.sub.2 wash (2 times)                                                                      0.5                                              2    45% trifluoroacetic acid (TFA) +                                                                      0.5                                                   5% 1,2-ethanedithiol in CH.sub.2 Cl.sub.2 (1 time)                       3    45% trifluoroacetic acid (TFA) +                                                                      20.0                                                  5% 1,2-ethanedithiol in CH.sub.2 Cl.sub.2 (1 time)                       4    CH.sub.2 Cl.sub.2 wash (3 times)                                                                      0.5                                              5    CH.sub.3 OH wash (2 times)                                                                            0.5                                              6    10% triethylamine (Et.sub.3 N) in CH.sub.2 Cl.sub.2                                                   0.5                                                   neutralization (2 times)                                                 7    CH.sub.3 OH wash (2 times)                                                                            0.5                                              8    10% triethylamine (Et.sub.3 N) in CH.sub.2 Cl.sub.2                                                   0.5                                                   neutralization (2 times)                                                 9    CH.sub.3 OH wash (2 times)                                                                            0.5                                             10    CH.sub.2 Cl.sub.2 wash (2 times)                                                                      0.5                                             11    *Boc-amino acid (1 mmole/g resin)                                                                     120                                                   plus equivalent amount of                                                     dicyclohexylcarbodiimide (DCC) in                                             CH.sub.2 Cl.sub.2                                                       12    CH.sub.2 Cl.sub.2 wash (1 time)                                                                       0.5                                             13    50% dimethylformamide in CH.sub.2 Cl.sub.2                                                            0.5                                                   wash (2 times)                                                          14    10% triethylamine (Et.sub.3 N) in CH.sub.2 Cl.sub.2                                                   0.5                                                   wash (1 time)                                                           15    CH.sub.3 OH wash (2 times)                                                                            0.5                                             16    CH.sub.2 Cl.sub.2 wash (2 times)                                                                      0.5                                             17    25% acetic anhydride in CH.sub.2 Cl.sub.2                                                             20.0                                                  (2 ml/g resin)                                                          18    CH.sub.2 Cl.sub.2 wash (2 times)                                                                      0.5                                             19    CH.sub.3 OH wash (2 times)                                                                            0.5                                             ______________________________________                                         *For the coupling of Asn and Gln, an 1.136 molar excess of                    1hydroxybenzotriazole (HOBt) is included in this step.                   

Briefly, for the coupling reaction, one mmol. of BOC-protected aminoacid in methylene chloride is used per gram of resin, plus oneequivalent of 0.5 molar DCCI in methylene chloride or 30% DMF inmethylene chloride, for two hours. When Arg is being coupled, a mixtureof 10% DMF and methylene chloride is used. Bzl is used as the hydroxylside-chain protecting group for Ser and Thr. 2-chloro-benzyloxycarbonyl(2Cl-Z) is used as the protecting group for the Lys side chain. Tos isused to protect the guanidino group of Arg, and the Glu or Asp carboxylgroup is protected as the Bzl ester. The phenolic hydroxyl group of Tyris protected with 2,6-dichlorobenzyl. Asn and Gln are left unprotected.At the end of the synthesis, the following composition is obtained(X¹)Tyr(X²)-Cys(X⁴)-Lys(X⁷)-Asn-Gly-Gly-Phe-Phe-Leu-Arg(X⁶)-Ile-His(X⁹)-Pro-Asp(X³)gly-Arg(X⁶-Val-Asp(X³)-Gly-Val-Arg(X⁶)-Glu(X³)-Lys(X⁷)-Ser(X.sup.5)-Asp(X³)-Pro-His(X⁹)-Ile-Lys(X⁷)-Leu-Gln-Leu-Gln-Ala-Glu(X³)-Glu(X³)-Arg(X⁶)-Gly-Val-Val-Ser(X⁵)-Ile-Lys(X⁷)-Gly-Val-X¹⁰wherein X¹ is BOC, X² is 2,6 -dichlorobenzyl, X³ is benyzl ester, X⁴ isMeOBzl, X⁵ is Bzl, X⁶ is Tos X⁷ is 2Cl-Z, X⁹ is Tos and X¹⁰ is -NH-MBHAresin support.

After the final Tyr residue has been coupled to the resin, the BOC groupis removed with 45% TFA in CH₂ Cl₂ In order to cleave and deprotect theremaining protected peptide-resin, it is treated with 1.5 ml. anisole,0.25 ml. methylethylsulfide and 10 ml. hydrogen fluoride (HF) per gramof peptide-resin, at -20° C. for one-half hour and at 0° C. for one-halfhour. After elimination of the HF under high vacuum, the resin-peptideremainder is washed alternately with dry diethyl ether and chloroform,and the peptide is then extracted with degassed 2N aqueous acetic acid.Lyophilization of the acetic acid extract provides a white fluffymaterial.

The cleaved and deprotected peptide is then dissolved in 30% acetic acidand subjected to Sephadex G-50 fine gel filtration.

The peptide is then further purified by CM-32 carboxymethyl cellulose(Whatman) cation-exchange chromatography(1.8×18 cm., V_(bed) =50 ml.)using a concave gradient generated by dropping 1 L. of 0.4 M NH₄ OAc, pH6.5 into a mixing flask containing 400 ml. 0.01 M. NH₄₀ Ac, pH 4.5.Final purification is carried out using preparative HPLC on a Vydec C₄column using a 0.1% TFA and acetonitrile solvent system. Purificationdetails are generally set forth in Ling et al. Biochem. Biophys. Res.Commun. 95, 945 (1980). The chromatographic fractions are carefullymonitored by TLC, and only the fractions showing substantial purity arepooled.

The synthesis is repeated using a chloromethylated resin to produce thesame peptide having a free acid C-terminus, generally following theprocedure described in Biopolymers, 12, 2513-19 (1973) to link Val tothe chloromethylated resin.

EXAMPLE II

To determine the effectiveness of the bFGF fragment peptide to inhibitthe growth of endothelial cells, the peptide is tested under conditionsto measure its ability to modulate both basal cell growth andbFGF-simulated cell proliferation. A bioassay was employed of the typeset forth in detail in Gospodarowicz et al., J. Cell Biol., 122, 323-333(1985), using BAAE cells.

For each test, an initial cell density of between about 0.3-0.5×10⁴cells per well was established in 24-miniwell plates. After 6-8 hours,the cells in each well were treated with a challenge dose of bFGF in theabsence, or presence to a varying concentration, of a synthetic FGFantagonist. The precise treatment was repeated 48 hours later. On thefifth day, the cells were digested with trypsin, and the total number ofcells in each well was determined using a Coulter particle counter.Testing of the peptide bFGF(24-68)-NH₂ shows full antagonist activity toboth basal cell growth and to bFGF-stimulated cell growth, with cellpopulation being reduced by about 84% and about 92%, respectively, at aconcentration of about 100 μg/ml. Like results are obtained from thetesting of bFGF(24-68)-OH, with both peptides exhibiting an ID₅₀ ofabout 5 micromoles.

Testing is then carried out to determine the effect of the fragments ofbFGF on the binding of I¹²⁵ -bFGF to BHK cells, in order to determinethe interaction with the receptors of FGF target cells, and is alsocarried out to determine the binding of the fragments to [³ H]-heparin.bFGF(24-68)-NH₂, at a concentration of 100 μg/ml., reduces the amount ofradioactive bFGF bound to the cells by about 54% and shows strongaffinity to bind heparin.

EXAMPLE III

The synthesis of -bFGF(30-50)-NH₂ having the formula:H-Phe-Phe-Leu-Arg-Ile-His-Pro-Asp-Gly-Arg-Val-Asp-Gly-Val-Arg-Glu-Lys-Ser-Asp-Pro-Tyr-NH₂ is conducted in a stepwise manner using a Beckman 990synthesizer and an MBHA resin in the manner described in Example I. Thepeptide is judged to be substantially pure using TLC and HPLC. Testingin the manner set forth in Example II shows that the peptide has fullantagonist activity to both basal and bFGF-stimulated endothelial cellgrowth, reducing cell population by about 19% and about 16%,respectively.

EXAMPLE IV

The synthesis of bFGF(30-49)-NH₂ having the formula:H-Phe-Phe-Leu-Arg-Ile-His-Pro-Asp-Gly-Arg-Val-Asp-Gly-Val-Arg-Glu-Lys-Ser-Asp-Pro-NH₂ is conducted in a stepwisemanner using a Beckman 990 synthesizer and an MBHA resin in the mannerdescribed in Example I except that cyclohexyl instead of Bzl is used toprotect Asp and Glu. The peptide is judged to be substantially pureusing TLC and HPLC. Testing in the manner set forth in Example II showsthat the peptide has full antagonist activity to both basal andFGF-stimulated endothelial cell growth.

EXAMPLE IV A

The synthesis of bFGF(25-37)-NH₂ having the formula:H-Cys-Lys-Asn-Gly-Gly-Phe-Phe-Leu-Arg-Ile-His-Pro-Asp-NH₂ is conductedin a stepwise manner using a Beckman 990 synthesizer and an MBHA resinin the manner described in Example I. The peptide is judged to besubstantially pure using TLC and HPLC. Testing in the manner set forthin Example II shows that the peptide has some antagonist activity tobasal endothelial cell growth and has a fairly strong binding affinityfor heparin and a fair affinity for BHK cells.

EXAMPLE V

The synthesis of [Tyr²⁵ ]-bFGF(25-68)-NH₂ having the formula:H-Tyr-Lys-Asn-Gly-Gly-Phe-Phe-Leu-Arg-Ile-His-Pro-Asp-Gly-Arg-Val-Asp-Gly-Val-Arg-Glu-Lys-Ser-Asp-Pro-His-Ile-Lys-Leu-Gln-Leu-Gln-Ala-Glu-Glu-Arg-Gly-Val-Val-Ser-Ile-Lys-Gly-Val-NH₂is conducted in a stepwise manner using a Beckman 990 synthesizer and anMBHA resin in the manner described in Example I. The peptide is judgedto be substantially pure using TLC and HPLC. Testing in the manner setforth in Example II shows that the peptide has full antagonist activityto both basal and bFGF-stimulated endothelial cell growth, reducing cellpopulation by about 86% and about 95%, respectively, and that it has avery strong binding affinity for BHK cells and heparin.

EXAMPLE VI

The synthesis of [Tyr³⁰,50 ]-bFGF(30-50)-OH having the formula:H-Tyr-Phe-Leu-Arg-Ile-His-Pro-Asp-Gly-Arg-Val-Asp-Gly-Val-Arg-Glu-Lys-Ser-Asp-Pro-Tyr-OHis conducted in a stepwise manner using a Beckman 990 synthesizer and achloromethylated resin in the manner described hereinbefore. The peptideis judged to be substantially pure using TLC and HPLC. Testing in themanner set forth in Example II shows that the peptide has fullantagonist activity to both basal and bFGF-stimulated endothelial cellgrowth.

EXAMPLE VII

The synthesis of bFGF(32-53)-NH₂ having the formula:H-Leu-Arg-Ile-His-Pro-Asp-Gly-Arg-Val-Asp-Gly-Val-Arg-Glu-Lys-Ser-Asp-Pro-His-Ile-Lys-Leu-NH₂is conducted in a stepwise manner using a Beckman 990 synthesizer and anMBHA resin in the manner described in Example I. The peptide is judgedto be substantially pure using TLC and HPLC. Testing in the manner setforth in Example II shows that the peptide has weak antagonist activityto both basal and bFGF-stimulated endothelial cell growth.

EXAMPLE VIII

The synthesis of bFGF(32-39)-N₂ having the formulaH-Leu-Arg-Ile-His-Pro-Asp-Gly-Arg-NH₂ is conducted in a stepwise mannerusing a Beckman 990 synthesizer and an MBHA resin in the mannerdescribed in Example I. The peptide is judged to be substantially pureusing TLC and HPLC. Testing in the manner set forth in Example II showsthat the peptide has full antagonist activity to both basal andbFGF-stimulated endothelial cell growth, reducing cell population byabout 37% and about 11%, respectively.

EXAMPLE IX

The synthesis of bFGF(24-63)-NH₂ having the formula:H-Tyr-Cys-Lys-Asn-Gly-Gly-Phe-Phe-Leu-Arg-Ile-His-Pro-Asp-Gly-Arg-Val-Asp-Gly-Val-Arg-Glu-Lys-Ser-Asp-Pro-His-Ile-Lys-Leu-Gln-Leu-Gln-Ala-Glu-Glu-Arg-Gly-Val-Val-NH₂is conducted in a stepwise manner using a Beckman 990 synthesizer and aMBHA resin in the manner described in Example I. The peptide is judgedto be substantially pure using TLC and HPLC. Testing in the manner setforth in Example II shows that the peptide has full antagonist activityto both basal and bFGF-stimulated endothelial cell growth.

EXAMPLE X

The synthesis of [Ala⁴⁷ ]-bFGF(24-63)-NH₂ having the formula:H-Tyr-Cys-Lys-Asn-Gly-Gly-Phe-Phe-Leu-Arg-Ile-His-Pro-Asp-Gly-Arg-Val-Asp-Gly-Val-Arg-Glu-Lys-Ala-Asp-Pro-His-Ile-Lys-Leu-Gln-Leu-Gln-Ala-Glu-Glu-Arg-Gly-Val-Val-NH₂ is conducted in a stepwise manner usinga Beckman 990 synthesizer and an MBHA resin in the manner described inExample I. The peptide is judged to be substantially pure using TLC andHPLC. Testing in the manner set forth in Example II shows that thepeptide has full antagonist activity to both basal and bFGF-stimulatedendothelial cell growth.

EXAMPLE XI

The synthesis of [Sar⁴² ]-bFGF(36-68)-NH₂ having the formula:H-Pro-Asp-Gly-Arg-Val-Asp-Sar-Val-Arg-Glu-Lys-Ser-Asp-Pro-His-Ile-Lys-Leu-Gln-Leu-Gln-Ala-Glu-Glu-Arg-Gly-Val-Val-Ser-Ile-Lys-Gly-Val-NH₂is conducted in a stepwise manner using a Beckman 990 synthesizer and anMBHA resin in the manner described in Example I. The peptide is judgedto be substantially pure using TLC and HPLC. Testing in the manner setforth in Example II shows that the peptide has full antagonist activityto both basal and bFGF-stimulated endothelial cell growth.

EXAMPLE XII

The synthesis of [Ala⁴² ]-bFGF(36-68)-NH₂ having the formula:H-Pro-Asp-Gly-Arg-Val-Asp-Ala-Val-Arg-Glu-Lys-Ser-Asp-Pro-His-Ile-Lys-Leu-Gln-Leu-Gln-Ala-Glu-Glu-Arg-Gly-Val-Val-Ser-Ile-Lys-Gly-Val-NH₂is conducted in a stepwise manner using a Beckman 990 synthesizer and anMBHA resin in the manner described in Example I. The peptide is judgedto be substantially pure using TLC and HPLC. Testing in the manner setforth in Example II shows that the peptide has full antagonist activityto both basal and bFGF-stimulated endothelial cell growth.

EXAMPLE XIII

The synthesis of bFGF(35-50)-NH₂ having the formula:H-His-Pro-Asp-Gly-Arg-Val-Asp-Gly-Val-Arg-Glu-Lys-Ser-Asp-Pro-His-NH₂ isconducted in a stepwise manner using a Beckman 990 synthesizer and anMBHA resin in the manner described in Example I. The peptide is judgedto be substantially pure using TLC and HPLC. Testing in the manner setforth in Example II shows that the peptide has full antagonist activityto both basal and bFGF-stimulated endothelial cell growth.

EXAMPLE XIV

The synthesis of [Ala⁴², Thr⁴⁷ ]-bFGF(35-50-NH₂ having the formula:H-His-Pro-Asp-Gly-Arg-Val-Asp-Ala-Val-Arg-Glu-Lys-Thr-Asp-Pro-His-NH₂ isconducted in a stepwise manner using a Beckman 990 synthesizer and anMBHA resin in the manner described in Example I. The peptide is judgedto be substantially pure using TLC and HPLC. Testing in the manner setforth in Example II shows that the peptide has full antagonist activityto both basal and bFGF-stimulated endothelial cell growth.

EXAMPLE XV

The synthesis of bFGF(36-39)-NH₂ having the formula:H-Pro-Asp-Gly-Arg-NH₂ is conducted in a stepwise manner using a Beckman990 synthesizer and an MBHA resin in the manner described in Example I.The tetrapeptide is judged to be substantially pure using TLC and HPLC.Testing in the manner set forth in Example II shows that the peptide hasfull antagonist activity to both basal and bFGF-stimulated endothelialcell growth, reducing cell population by about 37% and about 54%,respectively. It has biological potency less than that of bFGF(24-68),exhibiting an ID₅₀ at between about 30 and 50 micromoles.

EXAMPLE XVI

The synthesis of bFGF(93-120)-NH₂ having the formula:H-Phe-Phe-Phe-Glu-Arg-Leu-Glu-Ser-Asn-Asn-Tyr-Asn-Thr-Tyr-Arg-Ser-Arg-Lys-Tyr-Ser-Ser-Trp-Tyr-Val-Ala-Leu-Lys-Arg-NH₂ is conducted in a stepwise manner using a Beckman 990synthesizer and an MBHA resin in the manner described in Example I. Thepeptide is judged to be substantially pure using TLC and HPLC. Testingin the manner set forth in Example II shows that the peptide has fullantagonist activity to both basal and bFGF-stimulated endothelial cellgrowth, that it binds to heparin, and that it inhibits the binding ofbFGF to BHK cells.

EXAMPLE XVII

The synthesis of bFGF(106-118)-NH₂ having the formula:H-Tyr-Arg-Ser-Arg-Lys-Tyr-Ser-Ser-Trp-Tyr-Val-Ala-Leu-NH₂ is conductedin a stepwise manner using a Beckman 990 synthesizer and an MBHA resinin the manner described in Example I. The peptide is judged to besubstantially pure using TLC and HPLC. Testing in the manner set forthin Example II shows that the peptide has partial antagonist activity inmitogenic assays and inhibits binding of bFGF to its receptor in BHKcells.

EXAMPLE XVIII

The synthesis of bFGF(103-146)-NH₂ having the formula:H-Tyr-Asn-Thr-Tyr-Arg-Ser-Arg-Lys-Tyr-Ser-Ser-Trp-Tyr-Val-Ala-Leu-Lys-Arg-Thr-Gly-Gln-Tyr-Lys-Leu-Gly-Pro-Lys-Thr-Gly-Pro-Gly-Gln-Lys-Ala-Ile-Leu-Phe-Leu-Pro-Met-Ser-Ala-Lys-Ser-NH₂is conducted in a stepwise manner using a Beckman 990 synthesizer and anMBHA resin in the manner described in Example I. The peptide is judgedto be substantially pure using TLC and HPLC. Testing in the manner setforth in Example II shows that the peptide very strongly inhibits FGFbinding to BHK cells and to heparin.

EXAMPLE XVIII A

The synthesis of bFGF(97-120)-NH₂ having the formula:H-Arg-Leu-Glu-Ser-Asn-Asn-Tyr-Asn-Thr-Tyr-Arg-Ser-Arg-Lys-Tyr-Ser-Ser-Trp-Tyr-Val-Ala-Leu-Lys-Arg-NH₂is conducted in a stepwise manner using a Beckman 990 synthesizer and anMBHA resin in the manner described in Example I. The peptide is judgedto be substantially pure using TLC and HPLC. Testing is carried outusing a culture of serum-starved 3T3 cells which are incubated for 24hours with the bFGF peptide fragment and a challenge dose of bFGF andthen incubated for 5 hours with radioactive [³ H]-thymidine to determinewhether the fragment will inhibit the incorporation of [³ H]-DNA in thecell line which will be indicative of its inhibiting cell growth. It isshown that the peptide exhibits very good inhibition of bFGF-inducedmitosis, and further testing shows that it very strongly inhibits bFGFbinding to BHK cells and that it binds itself to heparin.

EXAMPLE XVIII B

The synthesis of bFGF(100-120)-NH₂ having the formula:H-Ser-Asn-Asn-Tyr-Asn-Thr-Tyr-Arg-Ser-Arg-Lys-Tyr-Ser-Ser-Trp-Tyr-Val-Ala-Leu-Lys-Arg-NH₂is conducted in a stepwise manner using a Beckman 990 synthesizer and anMBHA resin in the manner described in Example I. The peptide is judgedto be substantially pure using TLC and HPLC. Testing is carried outusing a culture of serum-starved 3T3 cells which are incubated for 24hours with the bFGF peptide fragment and a challenge dose of bFGF andthen incubated for 5 hours with radioactive [³ H]-thymidine to determinewhether the fragment will inhibit the incorporation of [³ H]-DNA in thecell line which will be indicative of its inhibiting cell growth. It isshown that the peptide exhibits very good inhibition of bFGF-inducedmitosis, and further testing shows that it very strongly inhibits bFGFbinding to BHK cells and that it binds itself to heparin.

EXAMPLE XVIII C

The synthesis of bFGF(103-120)-NH₂ having the formula:H-Tyr-Asn-Thr-Tyr-Arg-Ser-Arg-Lys-Tyr-Ser-Ser-Trp-Tyr-Val-Ala-Leu-Lys-Arg-NH₂is conducted in a stepwise manner using a Beckman 990 synthesizer and anMBHA resin in the manner described in Example I. The peptide is judgedto be substantially pure using TLC and HPLC. Testing is carried outusing a culture of serum-starved 3T3 cells which are incubated for 24hours with the bFGF peptide fragment and a challenge dose of bFGF andthen incubated for 5 hours with radioactive [³ H]-thymidine to determinewhether the fragment will inhibit the incorporation of [³ H]-DNA in thecell line which will be indicative of its inhibiting cell growth. It isshown that the peptide exhibits very good inhibition of bFGF-inducedcell mitosis; further testing shows that it very strongly inhibitsbinding of bFGF to BHK cells and that it binds to heparin.

EXAMPLE XVIII D

The synthesis of bFGF(106-120)-NH₂ having the formula:H-Tyr-Arg-Ser-Arg-Lys-Tyr-Ser-Ser-Trp-Tyr-Val-Ala-Leu-Lys-Arg-NH₂ isconducted in a stepwise manner using a Beckman 990 synthesizer and anMBHA resin in the manner described in Example I. The peptide is judgedto be substantially pure using TLC and HPLC. Testing is carried outusing a culture of serum-starved 3T3 cells which are incubated for 24hours with the bFGF peptide fragment and a challenge dose of bFGF andthen incubated for 5 hours with radioactive [³ H]-thymidine to determinewhether the fragment will inhibit the incorporation of [³ H]-DNA in thecell line which will be indicative of its inhibiting cell growth. It isshown that the peptide exhibits very good inhibition of bFGF-inducedcell mitosis; further testing shows that it very strongly inhibitsbinding of bFGF to BHK cells and that it binds to heparin.

EXAMPLE XVIII E

The synthesis of [Met¹¹⁴ ]-bFGF(106-120)-NH₂ having the formula:H-Tyr-Arg-Ser-Arg-Lys-Tyr-Ser-Ser-Met-Tyr-Val-Ala-Leu-Lys-Arg-NH₂ isconducted in a stepwise manner using a Beckman 990 synthesizer and anMBHA resin in the manner described in Example I. The peptide is judgedto be substantially pure using TLC and HPLC. Testing is carried out asdescribed in Example XVIIID. The peptide exhibits very good inhibitionof bFGF-induced cell mitosis and very strongly inhibits binding of bFGFto BHK cells.

EXAMPLE XVIII F

The synthesis of [Phe¹¹⁵ ]-bFGF(106-120)-NH₂ having the formula:H-Tyr-Arg-Ser-Arg-Lys-Tyr-Ser-Ser-Trp-Phe-Val-Ala-Leu-Lys-Arg-NH₂ isconducted in a stepwise manner using a Beckman 990 synthesizer and anMBHA resin in the manner described in Example I. The peptide is judgedto be substantially pure using TLC and HPLC. Testing is carried out asdescribed in Example XVIIID. The peptide exhibits very good inhibitionof bFGF-induced cell mitosis and very strongly inhibits binding of bFGFto BHK cells.

EXAMPLE XVIII G

The synthesis of bFGF(106-115)-NH₂ having the formula:H-Tyr-Arg-Ser-Arg-Lys-Tyr-Ser-Ser-Trp-Tyr-NH₂ is conducted in a stepwisemanner using a Beckman 990 synthesizer and an MBHA resin in the mannerdescribed in Example I. The peptide is judged to be substantially pureusing TLC and HPLC. Testing is carried out using a culture ofserum-starved 3T3 cells which are incubated for 24 hours with the bFGFpeptide fragment and a challenge dose of bFGF and then incubated for 5hours with radioactive [³ H]-thymidine to determine whether the fragmentwill inhibit the incorporation of [³ H]-DNA in the cell line which willbe indicative of its inhibiting cell growth. It is shown that thepeptide exhibits very good inhibition of bFGF-induced cell mitosis;further testing shows that it very strongly inhibits binding of bFGF toBHK cells and that it binds to heparin.

EXAMPLE XVIII H

The syntheses of the following compounds are conducted in a stepwisemanner using a Beckman 990 synthesizer and an MBHA resin in the mannerdescribed in Example I:

    ______________________________________                                                  bFGF(106-125)-NH.sub.2,                                                       bFGF(106-130)-NH.sub.2,                                                       bFGF(106-135)-NH.sub.2,                                                       bFGF(106-140)-NH.sub.2,                                                       bFGF(106-146)-NH.sub.2.                                             ______________________________________                                    

These peptides are each judged to be substantially pure using TLC andHPLC. Testing in the manner set forth in Example II shows that all thepeptides exhibit strong inhibition of bFGF binding to the receptor andinhibition of bFGF induced mitosis.

EXAMPLE XVIII J

The syntheses of the following compounds are conducted in a stepwisemanner using a Beckman 990 synthesizer and an MBHA resin in the mannerdescribed in Example I:

    ______________________________________                                               [D-Tyr.sup.106 ]-bFGF(106-120)-NH.sub.2,                                      [D-Arg.sup.107 ]-bFGF(106-120)-NH.sub.2,                                      [D-Ser.sup.108 ]-bFGF(106-120)-NH.sub.2,                                      [D-Arg.sup.109 ]-bFGF(106-120)-NH.sub.2,                                      [D-Lys.sup.110 ]-bFGF(106-120)-NH.sub.2,                                      [D-Tyr.sup.111 ]-bFGF(106-120)-NH.sub.2,                                      [D-Ser.sup.112 ]-bFGF(106-120)-NH.sub.2, and                                  [D-Ser.sup.113 ]-bFGF(106-120)-NH.sub.2.                               ______________________________________                                    

These peptides are each judged to be substantially pure using TLC andHPLC. Testing in the manner set forth in Example II shows inhibition ofbFGF binding to the FGF receptors of BHK cells nearly as strong as doesbFGF(106-120)-NH₂. Moreover, D-Ser¹¹³ showed inhibition to bFGF-inducedmitosis substantially as strong as bFGF(106-120)-NH₂.

EXAMPLE XVIII K

The synthesis of [Ala¹¹³ ]-bFGF(103-146)-NH₂ having the formula:H-Tyr-Asn-Thr-Tyr-Arg-Ser-Arg-Lys-Tyr-Ser-Ala-Trp-Tyr-Val-Ala-Leu-Lys-Arg-Thr-Gly-Gln-Tyr-Lys-Leu-Gly-Pro-Lys-Thr-Gly-Pro-Gly-Gln-Lys-Ala-Ile-Leu-Phe-Leu-Pro-Met-Ser-Ala-Lys-Ser-NH₂is conducted in a stepwise manner using a Beckman 990 synthesizer and anMBHA resin in the manner described in Example I. The peptide is judgedto be substantially pure using TLC and HPLC. Testing in the manner setforth in Example II shows that the peptide very strongly inhibits FGFbinding to BHK cells.

EXAMPLE XIX

Using conventional methods, described in CSH. supra., a syntheticbFGF-fragment gene is constructed having the following formula: ##STR2##

Synthesis of such a bFGF-fragment-encoding DNA chain is accomplished bysynthesizing oligonucleotides on an Applied Biosystems automaticsynthesizer with overlapping complementary sequences.

The overlapping oligonucleotides are fused to form a double-stranded DNAchain, gaps being filled in with DNA polymerase and with T4 ligase.Immediately 5, of the bFGF-fragment-encoding sequence in the sensestrand is provided an ATG start signal, which results in an extraneousmethionine being added to the N-terminus of the expressed polypeptide.Immediately 3' of the bFGF-fragment-encoding sequence is a stop signal.At the 5' end is a Eco RI overhang and at the 4' end is a Sal Ioverhang, whereby the synthetic DNA strand is directly insertable in theEco RI and Sal I site of the plasmid pUC8, described by Vieira et al.Gene 14, 259-268 (1982). The DNA strand is annealed into the pUC₈plasmid where it is under the control of the beta galactosidase promoterwith the ATG start signal and the Shine Delgarno sequence retained intheir natural orientation and association with the promoter.

The recombinant vector, designated bFGF(24-68), is transformed into theDH-1 strain of E. Coli by the calcium chloride procedure, CSH, supra.

The transformed E. Coli is cultured in L broth, and ampicillin-resistantstrains are selected. Because the DNA chain was inserted into theplasmid in an orientation which could be expected to lead to expressionof protein product of the DNA chain, the ampicillan-resistant coloniesare screened for reactivity with antiserum raised against bFGF. Thesecolonies are screened by the immunological method of Healfman et al.,Proc. Natl. Acad. Sci. USA 80, 31-35 (1983), and colonies reactingpositively with bFGF antibody are further characterized. The cells,following separation from their culture media, are lysed, and theirsupernatent obtained. Supernatent from these transformed cells isdetermined by RIA to be reactive with antibodies raised against bFGF.

100 ml. of cell supernatant is obtained, and the desired bFGF(24-68)fragment is purified as described above. Approximately 0.01 mg. ofbFGF(24-68), purified to upwards of 98% by weight of total protein, isproduced.

The biological activity of the synthetic bFGF fragment which containsthe extraneous N-terminal methionine residue, is tested for biologicalactivity with respect to ability to inhibit the growth of adult bovineaortic arch endothelial cells in culture, using an assay similar to thatdescribed in J. Cell Biol. 97, 1677-1685 (1983). Briefly, cells (atpassage 3-10) are seeded at a density of 2×10³ cells/dish on plastictissue culture dishes and exposed to Dulbecco's modified Eagle's medium(DMEM) supplemented with 10% calf serum. Test samples, at a dilutionranging from 10⁻¹ to 10⁻³, are added on day 0 and day 2 to the dishes.On day 4, triplicate dishes are trypsinized and counted in a Coultercounter. Background levels are ordinarily 10⁵ cells/dish, while thoseexposed to specified varying concentrations of the FGF antagonistcontain as few as 10⁴ cells/dish. For a potency assay, a log responsecurve is established. For this purpose, 10 microliter-aliquots of adilution (ranging from 10⁻¹ to 10⁻⁵) of the original solution made in0.5% bovine serum albumin (BSA)/DMEM are added in triplicate.

The superfluous N-terminal residue is removable by partial chemicaldigestion with cyanogen bromide or phenyl isothiocyanate followed bytreatment with a strong anhydrous acid, such as trifluoroacetic acid.After subjection to such cyanogen bromide treatment, the bFGF fragmentcontinues to substantially reduce the total number of cells present perdish.

EXAMPLE XX

A plasmid, following amplification in one of the bFGF-fragment producingE. Coli clones of Example XIX, is isolated and cleaved with Eco RI andSal I. This digested plasmid is electrophoresed on an agarose gelallowing for the separation and recovery of the amplified bFGF fragmentinsert. The insert is inserted into the plasmid pYEp, a shuttle vectorwhich can be used to transform both E. Coli and Saccharomyces cerevisiaeyeast. Insertion of the synthetic DNA chain at this point assures thatthe DNA sequence is under the control of a promoter, in proper readingframe from an ATG signal and properly spaced relative to a cap site. Theshuttle vector is used to transform URA3, a strain of S. cerevisiaeyeast from which the oratate monophophate decarboxylase gene is deleted.

The transformed yeast is grown in medium to attain log growth. The yeastis separated from its culture medium, and cell lysates are prepared.Pooled cell lysates are determined by RIA to be reactive with antibodyraised against bFGF, demonstrating that a peptide containing bFGFpeptide segments is expressed within the yeast cells.

The invention provides polypeptides which are biologically activeantagonists of both basic FGF and acidic FGF, because both have beenshown to act upon the same receptors, and should be available forbiological and therapeutic use. The production of longer bFGF fragmentscan be carried out in both prokaryotic and eukaryotic cell lines. Whilesuch synthesis is easily demonstrated using either bacteria or yeastcell lines, the synthetic genes should be insertable for expression incells of higher animals, such as mammalian tumor cells. Such mammaliancells may be grown, for example, as peritoneal tumors in host animals,and the desired bFGF fragments suitably harvested therefrom. The shorterbFGF fragments can simply be made by solid-phase or other coupling-typesynthesis.

Although the above examples demonstrate that bFGF-fragments can besynthesized through recombinant DNA techniques, the examples do notpurport to have maximized production. It is expected that subsequentselection of more efficient cloning vectors and host cell lines willincrease the yield of bFGF fragments. Known gene amplificationtechniques for both eukaryotic and prokaryotic cells may be used toincrease production. Secretion of the gene-encoded polypeptide from thehost cell line into the culture medium is also considered to be animportant factor in obtaining synthetic bFGF fragments in largequantities.

Brain and pituitary basic FGF preparations, as reported earlier, aremitogenic for a wide variety of normal diploid cultured cells derivedfrom tissue originating from the primary or secondary mesenchyme, aswell as from neuroectoderm. These include rabbit chondrocytes, bovinegranulosa and adrenal cortex cells, bovine corneal endothelial cells,capillary endothelial cells derived from bovine adrenal cortex and humanumbilical endothelial cells. FGF antagonists are useful biologicalmaterials for regulating in vitro growth of cultured cell lines and areexpected to also function in this manner when administered in vivolocally and otherwise. Accordingly, FGF antagonist peptides have manypotential therapeutic applications such as the treatment ofvasoproliferative diseases of the eye, e.g. diabetic retinopathies, ofproliferative diseases of the kidney, e.g. glomerulonephritis, ofcertain tumors, e.g. chondrosarcoma, and adrenal vascularization, aswell as inhibiting neovascularization of solid tumors in formation, andof other similar infirmities.

Because it appears that the growth of human melanomas and othermelanocytes is promoted by bFGF, the FGF antagonists should be effectiveto combat the growth of these cells and the growth promotion of certainrelated oncogenes, such as hst/KS3. More specifically, it is found thatbFGF antagonists, in the presence of heparin, inhibit the response ofmelanocytes to the transforming oncogene protein KS3. It is expectedthat these peptides will also be antagonists to other of the FGF familyof peptides, such as FGF-5.

Synthetic FGF antagonists or the nontoxic salts thereof, combined with apharmaceutically acceptable carrier to form a pharmaceuticalcomposition, may be administered to mammals, including humans, eitherintravenously, subcutaneously, intramuscularly or orally. The requireddosage will vary with the particular condition being treated, with theseverity of the condition and with the duration of desired treatment.

Such peptides are often administered in the form of pharmaceuticallyacceptable nontoxic salts, such as acid addition salts or metalcomplexes, e.g., with zinc, iron or the like (which are considered assalts for purposes of this application). Illustrative of such acidaddition salts are hydrochloride, hydrobromide, sulphate, phosphate,maleate, acetate, citrate, benzoate, succinate, malate, ascorbate,tartrate and the like. If the active ingredient is to be administered intablet form, the tablet may contain a binder, such as tragacanth, cornstarch or gelatin; a disintegrating agent, such as alginic acid; and alubricant, such as magnesium stearate. If administration in liquid formis desired, sweetening and/or flavoring may be used, and intravenousadministration in isotonic saline, phosphate buffer solutions or thelike may be effected.

The peptides should be administered under the guidance of a physician,and pharmaceutical compositions will usually contain the peptide inconjunction with a conventional, pharmaceutically-acceptable carrier.

Although the invention has been described with regard to its preferredembodiments, which constitute the best mode presently known to theinventors, it should be understood that various changes andmodifications as would be obvious to one having the ordinary skill inthis art may be made without departing from the scope of the inventionwhich is set forth in the claims appended hereto. For example, Thr canbe substituted for Ser in what would be position 112 of the bFGFmolecule, and Ser can be substituted for Pro in position 128, as thesedifferences appear in the highly homologous human bFGF molecule. As alsoindicated hereinbefore, Met can be substituted for Trp in the114-position, Phe can be substituted for Tyr in the 115-position, andAla can be substituted for Ser in the 113-position. Other similarsubstitutions as would be obvious to one skilled in peptide chemistrymay be made to provide equivalent FGF antagonists without departing fromthe scope of the invention. Extensions which do not change the FGFantagonist peptide into an FGF partial agonist having substantialagonist activity can be added to either or both termini, so long as theydo not significantly lessen its biological potency as an FGF antagonist,and such polypeptides are considered to be equivalents of thosedisclosed. For example, the residue Tyr can be added at either terminusof a synthetic FGF antagonist without substantially affecting thebiological potency of that particular antagonist. Inasmuch as thefunction of the peptide is primarily one of binding, it is the sequencethat is most important, and the C-terminus can be free acid, amide orsome equivalent moiety.

Specific features of the invention are emphasized in the claims whichfollow.

What is claimed is:
 1. A peptide of the formula:H-Tyr-Cys-Lys-Asn-Gly-Gly-Phe-Phe-Leu-Arg-Ile-His-Pro-Asp-Gly-Arg-Val-Asp-R₄₂-Val-Arg-Glu-Lys-R₄₇-Asp-Pro-His-Ile-Lys-Leu-Gln-Leu-Gln-Ala-Glu-Glu-Arg-Gly-Val-Val-Ser-Ile-Lys-Gly-Val-Y,wherein Y is OH or NH₂, R₄₂ is Gly, Ala or Sar and R₄₇ is Ser, Ala orThr or an N-terminally sequentially shortened fragment thereof or aC-terminally sequentially shortened fragment thereof or an N-terminallyand C-terminally sequentially shortened fragment thereof, which fragmentcontains the sequence Pro-Asp-Gly-Arg.
 2. A peptide according to claim 1wherein twelve residues in sequence beginning at the N-terminus aredeleted.
 3. A peptide according to claim 1 wherein twenty-nine residuesin sequence beginning at the C-terminus are deleted.
 4. A peptideaccording to claim 2 wherein twenty-nine residues in sequence beginningat the C-terminus are deleted.
 5. A peptide according to claim 1 whereinR₄₂ is Gly.
 6. A peptide according to claim 5 wherein R₄₇ in Ser. 7.H-Tyr-Cys-Lys-Asn-Gly-Gly-Phe-Phe-Leu-Arg-Ile-His-Pro-Asp-Gly-Arg-Val-Asp-Gly-Val-Arg-Glu-Lys-Ser-Asp-Pro-His-Ile-Lys-Leu-Gln-Leu-Gln-Ala-Glu-Glu-Arg-Gly-Val-Val-Ser-Ile-Lys-Gly-Val-NH₂.